In normal aging of humans and, to a much greater extent, in Alzheimer;s disease (AD) and Down's Syndrome, extracellular deposits of amyloid accumulate in senile plaques and blood vessel walls. Since the amyloid is among the first markers of aging in humans, we hypothesize here that the abnormal deposition of amyloid is detrimental to its surroundings, causes the neuritic response, and possibly, the tangle formation, synaptic and neuronal cell loss and finally cognitive deficits. One of the amyloid components is the beta-protein, a 4Kd fragment of a much larger protein precursor. We believe that an abnormal proteolytic degradation of the beta-protein precursor (betaPP) must occur in order to generate very hydrophobic fragments which can aggregate and precipitate as amyloid. A hint for an abnormal processing of the beta-PP comes from the second component found very tightly associated with the beta-protein, the serine protease inhibitor, alpha-1-antichymotrypsin (ACT). Finding ACT in the amyloid led us search for proteases which may be involved in the proteolytic degradation of the betaPP, we identified a protease fraction able to cleave at the peptides made according to known sequences of the betaPP, we identified a protease fraction able to cleave at the N-terminus of the beta-protein and are looking for a second protease that normally cleaves in the middle of the beta-protein. Here, we propose to study the appearance of amyloid in the aging and Ad brain and to correlate its formation with the presence of our proteases. In order to accomplish this goal, we need to purify the proteases to homogeneity, to sequence them and to produce monoclonal and polyclonal antibodies to them. The antibodies will provide tools for determining the distribution of the enzymes in brain and to clone their genes by screening expression cDNA libraries. The cloned cDNAs can be then sequenced and also used as probes on RNA blots to establish whether the proteases expression changes with increasing age or in various parts of the brain, especially those involved in amyloid deposits. The cloned cDNAs will then be used to identify the cells producing the enzymes by in situ hybridization and to localize the genes on the human chromosome. If the genes reside on chromosomes 21 or 19, the two possible locations for a familial Alzheimer's disease gene (FAD), we will perform linkage analysis using the proteases cDNAs to determine if either one of the genes coding for our proteases is the FAD gene. The reactive astrocytes are a good candidate for amyloidogenesis, either by providing their own betaPP or by means of secreting proteases and inhibitors involved in betaPP degradation. Here we will investigate the role of astrocytes in amyloid formation by using primary rat astrocytes transfected with human genes such as betaPP, ACT and the proteases, or by using human astrocytoma or glioblastoma cell lines. If we obtain an experimental system that can produce amyloid in cell culture, it will become a testable model for drug intervention in the hope of preventing beta-protein deposition, neuritic degeneration, neuronal cell loss and cognitive dysfunction, all seen in aging and Alzheimer's disease.